A zebrafish genetic toolkit to understand development

Development is complex business – from the moment a sperm fertilises an egg, a cascade of biological processes is set in motion. Small changes in this cascade can cause a number of different developmental conditions, and so trying to tease apart the stages is important to help find the causes and highlight potential treatment options. Here, Vanessa Chong discusses a new method the Sauka-Spengler Lab has developed to understand the nuts and bolts that regulate these developmental changes in a very special set of cells.

The neural crest is a population of cells in the vertebrate embryo, which have stem cell-like properties. This means that these cells can travel around the embryo and give rise to many different types of cells. These include the cells that make up the bones in the face, pigment cells, sensory neurons and the part of the nervous system that regulates the gut. Due to the diverse potential of neural crest cells, problems with their development lead to a range of different diseases, collectively known as neurocristopathies. Neurocristopathies include: CHARGE syndrome (features include a hole in the eye, heart defects, nose and ear defects, deafness, stunted growth and genital abnormalities); Hirschsprung’s disease (a genetic disorder affecting normal functioning of the colon) and Waardenburg syndrome (mainly characterised by deafness).

Understanding neural crest cellular identity is crucial to discovering potential points where processes can go wrong, leading to disease and developmental defects. Animals, including humans, are made up from hundreds of different cell types, but each cell has the same ‘black box’ of genetic information in their DNA. The key feature that makes one cell different from another is which specific bits of the genetic information (“genes”) are being read (“transcribed”) and used (“expressed”) at a given time and space.

For decades, scientists have discovered numerous groups of genes, which contain information to make proteins – biological molecules that orchestrate cellular function. Different cells produce different proteins at different times, forming a unique molecular signature for each cell type. So, what is the molecular signature of a neural crest cell? We created a new method the find out!

DNA that contains information to produce proteins only makes up ~1.5% of our genome. But what role does the rest – the so-called “junk” DNA – have? To get to the bottom of this we created a new technique called Biotagging. We found that in neural crest cells, the non-protein-coding regions are also active and contribute to neural crest identity. This finding is important because up until now, neural crest identity was based on the expression of genes that code for proteins called transcription factors.

So how did we discover this extra layer of complexity? We looked at non-protein-coding regions by genetically modifying zebrafish – a common tropical fish, frequently used in genetic studies because they lay lots of eggs at once, which can be easily accessed for biological experiments. These modified fish produced two key components of our Biotagging system. In one set of zebrafish, all the cell nuclei (the compartment in the cell where genetic information is kept) are tagged with a special protein tag, called “Avi”. In the second set of zebrafish, only the neural crest cells produce a protein that can add a molecular mark, called “biotin” onto the Avi-tag – a process called in vivo biotinylation. Breeding these two lines produces a subset of offspring that have neural crest cells with biotinylating ability, AND Avi-tagged nuclei. We then use special magnetic sticky beads that only recognise the Avi-tagged nuclei with biotin “marks”, which allows us to pick out the neural crest nuclei and leave behind nuclei from other cells.

This new system allows us to delve into the contents of the neural crest nuclei in detail, as we can watch a snapshot of different parts of the DNA being used, in real-time. This led to the finding that a cohort of non-protein-coding regions of DNA in the neural crest may be important for driving development of these special cells.

What next? We have just shown that there is more than meets the eye in trying to work out the identity of a neural crest cell. We hope to continue to shed light on the potential importance of transcribed non-coding elements, something that has so far been overlooked in neural crest biology. The Biotagging approach is designed to be flexible and not just limited to neural crest research – so far it has also been used to study the innate immune response to melanoma, heart regeneration and early blood development.